Project Summary Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Cells employ multiple repair and signaling responses to replication stress depending on the type, persistence, and location of the problem. We have employed proteomic and genetic approaches to understand how cells overcome replication stress. These analyses identified several new replication stress response proteins including RADX. RADX binds single-stranded DNA and prevents replication fork cleavage by endonucleases. We hypothesize that it regulates the processes of replication fork reversal and fork protection through its single-stranded DNA binding activity. We will test this hypothesis and more generally characterize mechanisms that regulate fork reversal and protection using biochemical and genetic approaches. Since cancer cells have elevated levels of replication stress and many cancer therapeutics work by interfering with DNA replication, these studies will also generate discoveries that may be translated into the cancer clinic.